CN108760727A - It is used as the purposes of catalyst as class peroxidase containing hydrionic small molecule - Google Patents
It is used as the purposes of catalyst as class peroxidase containing hydrionic small molecule Download PDFInfo
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- CN108760727A CN108760727A CN201810324001.XA CN201810324001A CN108760727A CN 108760727 A CN108760727 A CN 108760727A CN 201810324001 A CN201810324001 A CN 201810324001A CN 108760727 A CN108760727 A CN 108760727A
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- small molecule
- acid
- hydrogen peroxide
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- containing hydrionic
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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Abstract
The invention discloses the purposes for being used as catalyst as class peroxidase containing hydrionic small molecule.Present invention firstly discloses have class peroxidase activity containing hydrionic small molecule.The molecular structure containing hydrionic small molecule of the present invention is clear, is easy to get, stability is fine, and cheap, is easily commercially exploited.Based on the new application, the present invention also successfully constructs the new method of detection hydrogen peroxide, and is successfully applied in the test paper of detection hydrogen peroxide.The class peroxidase activity based on small molecule of the present invention can also be applied to the every field such as food, environment and health, there is extensive and good application prospect.
Description
Technical field
The invention belongs to analogue enztme fields, more particularly to contain hydrionic small molecule and be used as catalysis as class peroxidase
The purposes of agent.
Background technology
Enzyme is biocatalyst, closely related with vital movement.Enzyme is made of protein, generally unstable to acid, alkali, heat
It is fixed, it is easy inactivation denaturation, which has limited their extensive uses.Native enzyme is imitated to the molecular recognition of substrate and efficient catalytic
Analogue enztme research is a meaningful research field, and the important topic in many fields over the past decade.Horseradish peroxide
Compound enzyme is a kind of common toolenzyme, therefore, is had very important significance to the research and development tool of Mimetic Peroxidase.
Invention content
The shortcomings that it is an object of the invention to overcome the prior art and deficiency, are provided containing hydrionic small molecule as class mistake
Oxide enzyme is used as the purposes of catalyst.
Another object of the present invention is to provide one kind based on containing hydrionic small molecule measurement content of hydrogen peroxide or life
The method of object molecule content.
It is still another object of the present invention to provide the new opplications containing hydrionic small molecule.
The purpose of the invention is achieved by the following technical solution：It is used as class peroxidase containing hydrionic small molecule
The purposes of catalyst.
Described is hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, chlorine containing hydrionic small molecule
Acid, perchloric acid, hypochlorous acid, sulfurous acid, citric acid, oxalic acid, formic acid, propionic acid, butyric acid etc..The class peroxidase refers to
Show the substance of the catalytic activity of peroxidase.Specifically, class Catalyzed Synthesis By Peroxidase redox reaction of the invention,
And using peroxide as electron acceptor, to oxidation substrates.
The substrate is preferably peroxidase substrate, and under the conditions of existing for peroxidase substrate, described contains
Hydrionic small molecule can be used as catalyst, the reaction of catalyzing hydrogen peroxide and peroxidase substrate to lead to peroxide
Zymolyte produces coloured or luminescent products, can be surveyed to content of hydrogen peroxide by measuring the coloured or luminescent products of production
It is fixed.
The hydrogen peroxide can be generated by enzymatic reaction.
When hydrogen peroxide is generated by enzymatic reaction, refer to by biomolecule under specific protein enzyme effect, certain
Under the conditions of the hydrogen peroxide that generates.Therefore, under the conditions of existing for peroxidase substrate, described contains hydrionic small molecule
Catalyst, the reaction of catalyzing hydrogen peroxide and peroxidase substrate can be used as to cause peroxidase substrate production coloured
Or luminescent products, by measuring the coloured or luminescent products of production to be measured to the biomolecule content.
The biomolecule is preferably one kind or at least two in glucose, uric acid and cholesterol.
The specific proteases are preferably oxidizing ferment；Further preferably glucose oxidase, uricase, cholesterol oxygen
Change one kind or at least two in enzyme.
The peroxidase substrate is preferably 3,3,5,5- tetramethyl benzidines (TMB), 2,2- connection (the 3- second of nitrogen-two
Base-benzothiazole -6- sulfonic acid) di-ammonium salts (ABTS), one kind in o-phenylenediamine or at least two.
The certain condition can be 5~8,25~40 DEG C of pH.
The condition of the reaction of hydrogen peroxide and the peroxidase substrate is preferably 0~100 DEG C；More preferably 10~
40℃。
A method of content of hydrogen peroxide being measured using containing hydrionic small molecule, is based on containing hydrionic small molecule
It is used as the purposes realization of catalyst as class peroxidase, includes the following steps：It is catalysis to contain hydrionic small molecule
Agent makes the reaction of hydrogen peroxide and peroxidase substrate, generates coloring matter or luminescent substance, measures the coloring matter of generation
Or luminescent substance, so that it is determined that the content of hydrogen peroxide.
A method of biomolecule content being measured using containing hydrionic small molecule, is included the following steps：In specific egg
So that biomolecule oxidation is generated hydrogen peroxide under white enzymatic, hydrogen peroxide is measured using containing hydrionic small molecule according to above-mentioned
The method of content measures content of hydrogen peroxide, so that it is determined that biomolecule content.
The application containing hydrionic small molecule in field of biological detection or field of biosensors.
The application in field of biological detection can be the application in preparing test paper, reagent, kit；Institute
The test paper stated, reagent, kit detection object be preferably one kind or more in hydrogen peroxide, glucose, uric acid, cholesterol
Kind.
The biosensor is preferably hydrogen peroxide, glucose, uric acid or cholesterol sensor.
The present invention has the following advantages and effects with respect to the prior art：
1. the present invention is based on inventors be surprised to learn that there is class peroxidase activity containing hydrionic small molecule, provide
It is used as the new application of catalyst as class peroxidase containing hydrionic small molecule.Molecule knot containing hydrionic small molecule
Structure is clear, is easy to get, insensitive to heat, and stability is fine, and cheap, is easily commercially exploited；And it can be in short-term
Interior realization catalysis reaction.
2. the present invention utilizes the class peroxidase activity containing hydrionic small molecule, successfully constructs and detected
The new method of hydrogen oxide, and it is prepared for the test paper of detection hydrogen peroxide.
3. the present invention's can also be applied to food, environment and health etc. respectively based on hydrionic class peroxidase activity
A field, is with a wide range of applications.
Description of the drawings
Fig. 1 be the various concentration of embodiment 1 micromolecule catalyst phosphoric acid at ambient temperature catalyzing hydrogen peroxide with
The colour developing result photo figure of ABTS reactions；Wherein, concentration from left to right be followed successively by 1mol/L, 0.1mol/L, 0.01mol/L,
0.001mol/L、0.0001mol/L、0.00001mol/L。
Fig. 2 be the various concentration of embodiment 2 micromolecule catalyst hydrochloric acid at ambient temperature catalyzing hydrogen peroxide with
The colour developing result photo figure of ABTS reactions；Wherein, concentration from left to right be followed successively by 1mol/L, 0.1mol/L, 0.01mol/L,
0.001mol/L、0.0001mol/L、0.00001mol/L。
Fig. 3 be the various concentration of embodiment 3 micromolecule catalyst nitric acid at ambient temperature catalyzing hydrogen peroxide with
The colour developing result photo figure of ABTS reactions；Wherein, concentration from left to right be followed successively by 1mol/L, 0.1mol/L, 0.01mol/L,
0.001mol/L、0.0001mol/L、0.00001mol/L。
Fig. 4 be the various concentration of embodiment 4 micromolecule catalyst sulfuric acid at ambient temperature catalyzing hydrogen peroxide with
The colour developing result photo figure of ABTS reactions；Wherein, concentration from left to right be followed successively by 1mol/L, 0.1mol/L, 0.01mol/L,
0.001mol/L、0.0001mol/L、0.00001mol/L。
Fig. 5 be the various concentration of embodiment 5 micromolecule catalyst formic acid at ambient temperature catalyzing hydrogen peroxide with
The colour developing result photo figure of ABTS reactions；Wherein, concentration from left to right be followed successively by 1mol/L, 0.1mol/L, 0.01mol/L,
0.001mol/L、0.0001mol/L、0.00001mol/L。
Fig. 6 is that hydrogen peroxide Test paper made from embodiment 6 develops the color result photo under the hydrogen peroxide of various concentration
Figure.
Specific implementation mode
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The measurement of 1 peroxidase activity of embodiment
Concentrated phosphoric acid is diluted to respectively 1mol/L, 0.1mol/L, 0.01mol/L, 0.001mol/L, 0.0001mol/L,
The phosphoric acid solution of 0.00001mol/L concentration takes 0.1mL solution As (H containing 10mmol/L2O2, 10mmol/L 2,2- join nitrogen-two
(3- ethyls-benzothiazole -6- sulfonic acid) di-ammonium salts (ABTS)) it is added in the phosphoric acid solution of 0.9mL various concentrations, it reacts 1 minute.
The results are shown in Figure 1, which has good catalytic effect under suitable concentration.Color is deeper, indicates
Catalytic effect is better.
The measurement of 2 peroxidase activity of embodiment
Concentrated hydrochloric acid is diluted to respectively 1mol/L, 0.1mol/L, 0.01mol/L, 0.001mol/L, 0.0001mol/L,
The hydrochloric acid solution of 0.00001mol/L concentration takes 0.1mL solution As (H containing 10mmol/L2O2, 10mmol/L 2,2- join nitrogen-two
(3- ethyls-benzothiazole -6- sulfonic acid) di-ammonium salts (ABTS)) it is added in the hydrochloric acid solution of 0.9mL various concentrations, it reacts 1 minute.
The results are shown in Figure 2, which has good catalytic effect under suitable concentration.Color is deeper, indicates
Catalytic effect is better.
The measurement of 3 peroxidase activity of embodiment
Concentrated nitric acid is diluted to respectively 1mol/L, 0.1mol/L, 0.01mol/L, 0.001mol/L, 0.0001mol/L,
The salpeter solution of 0.00001mol/L concentration takes 0.1mL solution As (H containing 10mmol/L2O2, 10mmol/L 2,2- join nitrogen-two
(3- ethyls-benzothiazole -6- sulfonic acid) di-ammonium salts (ABTS)) it is added in the salpeter solution of 0.9mL various concentrations, it reacts 1 minute.
The results are shown in Figure 3, which has good catalytic effect under suitable concentration.Color is deeper, indicates
Catalytic effect is better.
The measurement of 4 peroxidase activity of embodiment
The concentrated sulfuric acid is diluted to respectively 1mol/L, 0.1mol/L, 0.01mol/L, 0.001mol/L, 0.0001mol/L,
The sulfuric acid solution of 0.00001mol/L concentration takes 0.1mL solution As (H containing 10mmol/L2O2, 10mmol/L 2,2- join nitrogen-two
(3- ethyls-benzothiazole -6- sulfonic acid) di-ammonium salts (ABTS)) it is added in the sulfuric acid solution of 0.9mL various concentrations, it reacts 1 minute.
The results are shown in Figure 4, which has good catalytic effect under suitable concentration.Color is deeper, indicates
Catalytic effect is better.
The measurement of 5 peroxidase activity of embodiment
Formic acid is diluted to respectively 1mol/L, 0.1mol/L, 0.01mol/L, 0.001mol/L, 0.0001mol/L,
The formic acid solution of 0.00001mol/L concentration takes 0.1mL solution As (H containing 10mmol/L2O2, 10mmol/L2,2- connection nitrogen-two (3-
Ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts (ABTS)) it is added in the formic acid solution of 0.9mL various concentrations, it reacts 1 minute.
The results are shown in Figure 5, which has good catalytic effect under suitable concentration.Color is deeper, indicates
Catalytic effect is better.
The preparation and application of 6 hydrogen peroxide Test paper of embodiment
(1) maceration extract is prepared：One, 1000mL beakers are taken, 40mL sodium alginate solns (a concentration of 50g/L) are added, are added
It is a concentration of that 40mL gelatin solutions (a concentration of 80g/L), addition 100mL is added in the Tween 80 solution of 10mL a concentration of 1% (v/v)
Formic acid solution, 10mL liquor kalii iodides (50g/L), 10g polyvinylpyrrolidones, the 0.5g brilliant blues of 1mol/L, mixes well, obtains
To maceration extract；
(2) impregnated paper：Step (1) prepared maceration extract is poured into glass dish, filter paper is impregnated, it will be extra with glass bar
Solution scrapes off, and is put into drying 30 minutes in 40 DEG C of convection ovens, is detected to get the hydrogen peroxide for pale blue test paper after doing
Test paper.
Various concentration hydrogen peroxide (0 μM, 50 μM, 100 μM, 300 μM, 500 μM, 700 μM, 1000 μM) to be measured is direct
It drops on obtained hydrogen peroxide Test paper, the reaction time is 1 minute.
The results are shown in Figure 6, and test paper can show different color changes, with the raising of concentration of hydrogen peroxide, color
There is pale blue, green, yellow to brown changes.
The preparation and application of 7 hydrogen peroxide Test paper of embodiment
(1) maceration extract is prepared：One, 1000mL beakers are taken, 40mL sodium alginate solns (a concentration of 50g/L) are added, are added
It is a concentration of that 40mL gelatin solutions (a concentration of 80g/L), addition 100mL is added in the Tween 80 solution of 10mL a concentration of 1% (v/v)
Phosphoric acid solution, 10mL liquor kalii iodides (50g/L), 10g polyvinylpyrrolidones, the 0.5g brilliant blues of 0.02mol/L, it is fully mixed
It is even, obtain maceration extract；
(2) impregnated paper：Step (1) prepared maceration extract is poured into glass dish, filter paper is impregnated, it will be extra with glass bar
Solution scrapes off, and is put into drying 30 minutes in 40 DEG C of convection ovens, is detected to get the hydrogen peroxide for pale blue test paper after doing
Test paper.
Various concentration hydrogen peroxide (0 μM, 50 μM, 100 μM, 300 μM, 500 μM, 700 μM, 1000 μM) to be measured is direct
It drops on obtained hydrogen peroxide Test paper, the reaction time is 1 minute.
As a result it is similar to embodiment 6, test paper can show different color changes, with the raising of concentration of hydrogen peroxide,
There is pale blue in color, and green, yellow to brown changes.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (10)
1. being used as the purposes of catalyst as class peroxidase containing hydrionic small molecule.
2. it is used as the purposes of catalyst as class peroxidase containing hydrionic small molecule according to claim 1, it is special
Sign is：It is described containing hydrionic small molecule be hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, chloric acid,
One kind in perchloric acid, hypochlorous acid, sulfurous acid, citric acid, oxalic acid, formic acid, propionic acid and butyric acid or at least two.
3. it is a kind of using the method for measuring content of hydrogen peroxide containing hydrionic small molecule, it is based on hydrogeneous described in claim 1
The small molecule of ion is used as the purposes realization of catalyst as class peroxidase, it is characterised in that includes the following steps：With
It is catalyst containing hydrionic small molecule, makes the reaction of hydrogen peroxide and peroxidase substrate, generate coloring matter or shines
Substance measures the coloring matter or luminescent substance of generation, so that it is determined that the content of hydrogen peroxide.
4. utilizing the method for measuring content of hydrogen peroxide containing hydrionic small molecule according to claim 3, it is characterised in that：
The hydrogen peroxide is generated by enzymatic reaction.
5. utilizing the method for measuring content of hydrogen peroxide containing hydrionic small molecule according to claim 3, it is characterised in that：
The peroxidase substrate is 3,3,5,5- tetramethyl benzidines, 2,2- connection (the 3- ethyls-benzothiazole -6- sulphurs of nitrogen-two
Acid) di-ammonium salts, one kind in o-phenylenediamine or at least two.
6. utilizing the method for measuring content of hydrogen peroxide containing hydrionic small molecule according to claim 3, it is characterised in that：
The temperature of the reaction is 0~100 DEG C.
7. a kind of utilizing the method for measuring biomolecule content containing hydrionic small molecule, it is characterised in that include the following steps：
Biomolecule oxidation is set to generate hydrogen peroxide under specific protein enzymatic, according to claim 3 using containing hydrionic
The method that small molecule measures content of hydrogen peroxide measures content of hydrogen peroxide, so that it is determined that biomolecule content.
8. utilizing the method for measuring biomolecule content containing hydrionic small molecule according to claim 7, it is characterised in that：
The biomolecule is one kind or at least two in glucose, uric acid and cholesterol；
The protease is oxidizing ferment.
9. the application containing hydrionic small molecule in field of biological detection or field of biosensors is to be based on claim 1
What the purposes for being used as catalyst as class peroxidase containing hydrionic small molecule was realized.
10. the answering in field of biological detection or field of biosensors containing hydrionic small molecule according to claim 9
With, it is characterised in that：
The application containing hydrionic small molecule in field of biological detection is answering in preparing test paper, reagent, kit
With；
The biosensor is hydrogen peroxide, glucose, uric acid or cholesterol sensor.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810324001.XA CN108760727A (en) | 2018-04-12 | 2018-04-12 | It is used as the purposes of catalyst as class peroxidase containing hydrionic small molecule |
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| CN201810324001.XA CN108760727A (en) | 2018-04-12 | 2018-04-12 | It is used as the purposes of catalyst as class peroxidase containing hydrionic small molecule |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109632780A (en) * | 2018-12-27 | 2019-04-16 | 广东轻工职业技术学院 | A kind of colorimetric method and kit detecting ATP |
| CN111537463A (en) * | 2020-04-15 | 2020-08-14 | 广东省第二人民医院（广东省卫生应急医院） | Method for quantitatively detecting uric acid in serum |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101387606A (en) * | 2008-08-01 | 2009-03-18 | 中国科学院长春应用化学研究所 | Method for detecting hydrogen peroxide or glucose based on enzyme simulation by ferroferric oxide magnetic nanometer particle |
| CN107831161A (en) * | 2017-12-06 | 2018-03-23 | 广东轻工职业技术学院 | Small molecule containing acetate is used as the new application of catalyst as class peroxidase |
-
2018
- 2018-04-12 CN CN201810324001.XA patent/CN108760727A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101387606A (en) * | 2008-08-01 | 2009-03-18 | 中国科学院长春应用化学研究所 | Method for detecting hydrogen peroxide or glucose based on enzyme simulation by ferroferric oxide magnetic nanometer particle |
| CN107831161A (en) * | 2017-12-06 | 2018-03-23 | 广东轻工职业技术学院 | Small molecule containing acetate is used as the new application of catalyst as class peroxidase |
Non-Patent Citations (2)
| Title |
|---|
| YX699313: "TMB和双氧水的显色", 《小木虫》 * |
| ZINCPENG: "TMB和双氧水可以直接反应吗", 《小木虫》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109632780A (en) * | 2018-12-27 | 2019-04-16 | 广东轻工职业技术学院 | A kind of colorimetric method and kit detecting ATP |
| CN109632780B (en) * | 2018-12-27 | 2021-04-06 | 广东轻工职业技术学院 | Colorimetric method and kit for detecting ATP |
| CN111537463A (en) * | 2020-04-15 | 2020-08-14 | 广东省第二人民医院（广东省卫生应急医院） | Method for quantitatively detecting uric acid in serum |
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